Aryn Gittis, Ph.D. - US grants

DESCRIPTION (provided by applicant): The basal ganglia are a highly conserved neural system that play a role in movement, learning, and cognition. The output of the basal ganglia is controlled by two neural pathways that either facilitate movement (direct pathway) or inhibit movement (indirect pathway). Dissruption in the balance of these two pathways underlies motor defecits observed in neurological diseases such as dystonia, Parkinson's disease, Tourette's syndrome, and Huntington's disease. Understanding how direct- and indirect-pathway circuits are coordinated at the cellular level is therefore of great importance and potential therapeutic value. Over the past decade, studies investigating the cellular organization of network function have converged upon classes of highly specialized neurons called inhibitory interneurons. The importance of these neurons in basal ganglia function is demonstrated by the fact that their loss in the striatum, the input nucleus of the basal ganglia, severely impairs motor function. Despite their importance, the basic role of interneurons in striatal processing remains poorly understood because they have been historically difficult to target for electrophysiological study. This proposal describes our use of a novel approach to study the role of inhibitory interneurons in the striatum, by using transgenic mouse lines to fluorescently label distinct cell types in the striatal circuit. This new technology enables direct testing of how inhibitory signaling affects neurons in the direct and indirect basal ganglia pathways for the first time. Using this approach, we will tests three hypotheses about inhibitory interneuron function in the striatum: (1) That different classes of interneurons play distinct roles in striatal processing (2) that striatal interneurons receive an important feedback signal that tunes striatal output and (3) that interneurons contribute to imbalances in striatal output (hyperactivation of the indirect-pathway) observed during Parkinson's disease. The experiments to test these hypotheses will utilize whole-cell recording techniques to study excitability and synaptic signaling of interneurons in acute brain slices. The slices will be made from transgenic mice where distinct cell types in the striatal circuit are fluorescently labeled, enabling discoveries about synaptic properties and connectivity to be placed in a systems-level context of circuit function. These results will yield insights into how synaptic plasticity or reorganization changes striatal output in PD and how this might contribute to circuit dysfunction in PD and other diseases of the basal ganglia.

Project Summary/Abstract The basal ganglia are a series of inter-connected brain nuclei that control voluntary movement through the coordination of two parallel neural circuits, the 'direct pathway' that facilitates movement and the 'indirect pathway' that suppresses movement. Imbalances between these two pathways are hypothesized to underlie movement disorders such as Parkinson's disease, Huntington's disease, Tourette syndrome, and dystonia. My long-term goal is to identify cellular mechanisms of direct and indirect pathway regulation to better understand the neuronal basis of motor control in both health and disease. Neuronal circuits in the striatum, the input nucleus of the basal ganglia, are particularly important in determining direct and indirect pathway activity, but their organizing principles remain poorly understood. The objective of this proposal is to identify the role of fast-spiking (FS) interneurons in regulating striatal output. In Aim 1, I will use a novel pharmacological approach to perform circuit-level and behavioral analyses of FS interneuron regulation of direct and indirect pathway activity. Because interneurons are powerful regulators of circuit function and are intimately involved in the pathology of several basal ganglia disorders, this pharmacological dissection of their role will provide new insights into Parkinson's disease and other disorders and may lead to new pharmaceutical treatments. In Aim 2-3, I will use optogenetics combined with slice and in vivo electrophysiology to challenge long-standing hypotheses about the organization of FS microcircuits. Long- standing theories about inputs to FS interneurons from the cortex and thalamus have shaped thinking about the organization of striatal microcircuits, but these dogmatic theories are based largely on experiments in other systems and have not been directly tested in the striatum. Using optogenetics in Aim 2, I will directly measure feedforward inhibition recruited by cortical and thalamic inputs to the striatum to directly challenge old ideas about the role of feedforward inhibition in striatal function and disease. In Aim 3, I will turn to FS inputs from the globus pallidus (GPe); although inputs from the GPe were identified anatomically over ten years ago, a detailed functional characterization of this projection has been sorely lacking. The identification of novel neural circuits that connect the GPe and striatum could challenge long-standing assumptions about information processing in the basal ganglia. Together, all of these experiments will advance our understanding of the neural circuitry that underlies motor control and potentially identify new targets for disease therapy.

In specialized brain areas called the basal ganglia, neurons generate certain rhythmic activity patterns during particular stages of motor processing. This rhythmicity becomes enhanced in disorders such as Parkinson's disease (PD), where pathological activity patterns include pronounced oscillations within certain frequency bands, increased synchrony, and phasic bursting. These patterns of amplified rhythmicity are thought to compromise functionality of basal ganglia networks and to contribute to motor impairments. The overarching goal of this work is to understand how cellular and synaptic changes that occur in PD render neural circuits in the basal ganglia more susceptible to rhythmic activity in disease. Specifically, this research will focus on an understudied neural circuit that is well positioned to influence rhythmicity throughout the basal ganglia and will help to identify potential cellular targets to disrupt pathological network activity in disease. A combination of in vivo and in vitro physiological approaches together with computational model development, simulation, and analysis will be used to identify biological features of this circuit that can generate and maintain rhythmicity and to explain how this network contributes to runaway rhythmicity in PD.

The external segment of the globus pallidus (GPe) is a central nucleus within the basal ganglia that has been strongly implicated in the onset and maintenance of rhythmic activity. Under normal conditions, neurons in the GPe fire tonically and independently, at rates of 10-80 Hz; after dopamine depletion, GPe neurons become highly synchronized and fire in rhythmic bursts. The GPe interacts with other basal ganglia nuclei through multiple circuits, one of which, a pallidostriatal circuit linking GPe with the striatum, has only recently been identified as a natural contributor to basal ganglia rhythmicity. A major goal of this work is the identification of features within the pallidostriatal circuit that generate and maintain rhythmicity and may underlie runaway rhythmicity in pathological conditions. Achievement of this aim will help in the location of targets that can be modulated to disrupt the pathological amplification or propagation of basal ganglia rhythmicity. These results will be achieved through an approach that integrates in vivo and in vitro physiological experiments with computational model development, simulation, and analysis.

? DESCRIPTION (provided by applicant): Parkinson's disease (PD) is a movement disorder whose hallmark motor symptoms arise due to loss of dopaminergic innervation of the striatum. Motor symptoms include slowed movement, decreased coordination, and gait abnormalities. These symptoms typically do not present until dopamine levels in the striatum have been reduced by 70-80%. Clinically, this means that patients who seek medical help at the onset of motor symptoms have likely been living with chronically low levels of dopamine for years. This early phase of the disease, when dopamine levels are pathologically low but motor symptoms have not yet presented, is called the prodromal phase, and is likely the optimal period in which to administer therapies. However, most research utilizing animal models of parkinsonian motor impairments investigates circuit dysfunction only in late stages of depletion, after severe motor dysfunction has already occurred. These animal models have greatly advanced our understanding of the synaptic and circuit-level changes present in massively dopamine depleted animals, but we still know very little about the progression of circuit dysfunction, or compensatory plasticity leading up to the appearance of motor impairments, an understanding that may be critical to halting disease progression before it becomes incurable. The primary goal of the proposed research is to study the progression of circuit dysfunction and compensation leading up to the appearance of motor deficits using a novel dopamine depletion paradigm where the onset and progression of dopamine loss can be tightly controlled. In Aim 1, we will conduct a nonbiased, high throughput screen for brain areas showing differential activation in mice gradually depleted over weeks to months, relative to acutely depleted mice. Brain areas showing differential activity in acutely vs. gradually depleted animals will identify potential sits of compensatory plasticity, providing a foundation for future studies of the cellular and synaptic mechanisms of network adaptations during prodromal PD. In Aim 2, we will validate our model by comparing patterns of brain activity in gradually depleted mice to those observed in an established genetic model of PD, Thy1-?Syn, where ~40% of dopamine is lost by age 14 months. Brain areas showing common changes across both models will reveal the most promising sites of disease-relevant plasticity. Finally, in Aim 3, we will use cutting-edge electrophysiological approaches to record neural activity in candidate brain areas over the duration of our gradual depletion paradigm. These experiments will identify neural correlates of network compensation leading up the appearance of motor deficits. Combined, these results will provide novel insights into the location and progression of compensatory plasticity during prodromal PD, and will lay the foundation for future studies of the cellular and synaptic basis of adaptive plasticity in disease.

Abstract A major challenge in the treatment of neurological diseases is the elaborate and diffuse nature of neural circuits, where physically proximal neurons are engaged in functionally different pathways. The ability to target neurons based on function, rather than location, is critical to improving treatments for disease. In Parkinson's disease, improved treatments have been driven by the discovery of cell type diversity in the striatum, providing access to functionally opposing circuits: the direct and indirect pathways. However, with the exception of neuronal diversity in the striatum, all other downstream nuclei in the basal ganglia are depicted as homogeneous relay nuclei, an oversimplification whose limits are increasingly apparent as techniques to study circuit function become more sophisticated. Recently, my lab has pioneered the use of transgenic mouse lines to subdivide neurons in the external globus pallidus (GPe) into subpopulations that differ in anatomy and electrophysiological properties. Leveraging tools to optogenetically manipulate these genetic subpopulations, we are now in position to discover their contributions to behavior. In a recent study, we found that optogenetic interventions targeted to particular subpopulations in the GPe (but not global stimulation of the entire nucleus) restores motor function in acutely dopamine depleted mice, and the effects persisted for hours after stimulation. This finding challenges long-standing models of circuit organization in the basal ganglia and has relevance for PD, where current interventions provide only transient relief of motor symptoms that rapidly return once stimulation stops. Experiments in this proposal will test the ability of GPe interventions to rescue movement in a chronic dopamine depletion model (Aim 1) and will elucidate the pathways through which GPe subpopulations mediate their effects (Aim 2). Aim 1, will use optogenetics and in vivo recordings to assess the impact of modulating genetically-defined neuronal subpopulations on local circuit dynamics in the GPe and their effects on behavior. Specifically, we will test the hypothesis that recovered movements following optogenetic stimulation are goal-directed and restore the ability of mice to seek out food, social interactions, and avoid anxiety-provoking environments. In Aim 2, we will use in vivo recordings, coupled with viral-assisted circuit mapping, to elucidate the pathways through which neuronal subpopulations in the GPe exert their prokinetic effects on movement. Our preliminary data suggest that therapeutic interventions share a common mechanism of reversing pathological firing patterns in the substantia nigra reticulata (SNr), the primary basal ganglia output nucleus in rodents. Our proposed experiments will determine whether this effect is mediated by direct projections of GPe neurons to the SNr, or whether it is mediated through a disynaptic pathway involving the subthalamic nucleus (STN). Combined, results from these studies will elucidate the pathways and circuit mechanisms responsible for long-lasting motor rescue in dopamine depleted mice and will revise long-standing models of indirect pathway dysfunction in disease.

Abstract    A major challenge in the treatment of neurological diseases is the elaborate and diffuse nature of neural  circuits, where physically proximal neurons are engaged in functionally different pathways. The ability to target  neurons based on function, rather than location, is critical to improving treatments for disease. In Parkinson?s  disease, improved treatments have been driven by the discovery of cell type diversity in the striatum, providing  access to functionally opposing circuits: the direct and indirect pathways. However, with the exception of  neuronal diversity in the striatum, all other downstream nuclei in the basal ganglia are depicted as  homogeneous relay nuclei, an oversimplification whose limits are increasingly apparent as techniques to study  circuit function become more sophisticated. Recently, my lab has pioneered the use of transgenic mouse lines  to subdivide neurons in the external globus pallidus (GPe) into subpopulations that differ in anatomy and  electrophysiological properties. Leveraging tools to optogenetically manipulate these genetic subpopulations,  we are now in position to discover their contributions to behavior. In preliminary studies, we found that  optogenetic interventions targeted to particular subpopulations in the GPe (but not global stimulation of the  entire nucleus) could restore motor function in dopamine depleted mice and the effects persisted for hours  after stimulation. This finding challenges long-­standing models of circuit organization in the basal ganglia and  has relevance for PD, where current interventions provide only transient relief of motor symptoms that rapidly  return once stimulation stops. Experiments in this proposal will identify which neuronal subpopulations in the  GPe are required to induce long-­lasting motor rescue (Aim 1) and will elucidate the pathways through which  they mediate their effects (Aim 2). Aim 1, will use optogenetics and in vivo recordings to assess the impact of  modulating genetically-­defined neuronal subpopulations on local circuit dynamics in the GPe and their effects  on behavior. Specifically, we will test the hypothesis that recovered movements following optogenetic  stimulation are goal-­directed and restore the ability of mice to seek out food, social interactions, and avoid  anxiety-­provoking environments. In Aim 2, we will use in vivo recordings, coupled with viral-­assisted circuit  mapping, to elucidate the pathways through which neuronal subpopulations in the GPe exert their prokinetic  effects on movement. Our preliminary data suggest that therapeutic interventions share a common mechanism  of reversing pathological firing patterns in the substantia nigra reticulata (SNr), the primary basal ganglia output  nucleus in rodents. Our proposed experiments will determine whether this effect is mediated by direct  projections of GPe neurons to the SNr, or whether it is mediated through a disynaptic pathway involving the  subthalamic nucleus (STN). Combined, results from these studies will elucidate the pathways and circuit  mechanisms responsible for long-­lasting motor rescue in dopamine depleted mice and will revise long-­standing  models of indirect pathway dysfunction in disease.       

Abstract Deep brain stimulation (DBS) is one of the most effective treatments for patients with advanced Parkinson's disease (PD). Delivery of high frequency electrical stimulation to the subthalamic nucleus (STN) ameliorates parkinsonian motor symptoms, often within seconds, but therapeutic effects wear off quickly if stimulation is stopped, often within minutes. This transient nature of symptomatic relief underscores the fact that existing DBS protocols mask symptoms but do not alleviate underlying circuit dysfunction. A modified DBS protocol, called coordinated reset (CR-DBS), has shown potential to provide long-lasting therapeutic benefits for days to weeks after stimulation, but this protocol has been slow to translate into widespread clinical use because (1) the multi- site, pseudorandom stimulation patterns required to implement it cannot be delivered with existing devices and (2) its mechanisms of action remain obscure, hindering insights into what parameters of CR-DBS should be tuned to ensure engagement of long-lasting effects. Recently, in a mouse model of PD, we discovered a cellular- based strategy to induce long-lasting motor recovery, by using optogenetics to target interventions to specific neuronal subpopulations in the external globus pallidus (GPe), an anatomical neighbor of the STN. Long-lasting motor rescue was induced by interventions that simultaneously increased the firing rates of GPe neurons enriched in parvalbumin (PV-GPe) and decreased the firing rates of GPe neurons enriched in lim homeobox 6 (Lhx6-GPe). Interestingly, at the physiological level, these cell-type specific interventions in the GPe converged upon a similar mechanism as CR-DBS, by ameliorating pathological patterns of neural activity in basal ganglia output nuclei that have been associated with parkinsonian motor deficits. This proposal will use knowledge gained from our discovery of long-lasting rescue through cell-type directed interventions in GPe to guide rational design and interrogation of human-applicable forms of DBS that may yield similarly long-lasting therapeutic benefit. Our experiments will test a novel, mechanistic hypothesis, based on supporting preliminary data, that the pattern of electrical DBS can be tuned to drive cell-type specific responses in the GPe that mirror those previously found to be sufficient to induce of long-lasting motor rescue with optogenetics. Experiments in Aim 1 will investigate the cellular mechanisms through which phasic stimulation in the STN evokes cell-type specific responses in the GPe (Aim 1.1) and use a machine learning approach to identify stimulation protocols that maximize this cell-type specific response (Aim 1.2). Experiments in Aim 2 will test the therapeutic efficacy of phasic stimulation protocols compared to conventional DBS, using behavioral and physiological assays in mouse (Aim 2.1) and primate (Aim 2.2) models of PD. Taken together, these experiments will advance our understanding of the fundamental differences between how conventional vs. phasic stimulation impacts the nervous system, with cell-type specific and synapse-specific resolution, and could provide novel therapeutic strategies that can be rapidly translated into humans.